Association of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with other macromolecules and cellular structures (e.g. Band 3, microtubules, triad junctions) has been frequently reported. The functional significance, if any, of these associations is unknown. A single amino acid change in the GAPDH of a mutant Chinese hamster ovary cell (FD1.3.25) results in an association of that protein with microtubules that cannot be disrupted by agents (e.g. high salt, ATP) that effect dissociation of normal GAPDH; this correlates with genetically dominant alterations in endocytosis and aberrant association of late endosomes with microtubules observed in FD1.3.25. FD1.3.25 is a heterozygote, with normal and mutated GAPDH alleles; based on steady- state RNA levels, the mutated form represents 25% of the total GAPDH; moreover, the enzyme is a tetramer; thus very little of the GAPDH from FD1.3.25 is of "purely mutant" form. In order to study the properties of the mutant enzyme, GAPDH CDNA bearing the FD1.3.25 mutation has been overexpressed in E. coli.